1. Field of the Invention
The invention is in the field of synthetic polycationic polymers, and the use of synthetic polycationic polymers as nucleic acid transfection agents and anionically charged bioactive agent delivery vehicles. In a particular embodiment the invention relates to polycationic polymers obtained from the copolymerization of multifunctional diprimary amines and dicarboxylic monosaccharides.
2. Background Art
In recent years gene therapy, i.e., the use of genetic materials for medical treatments, has received tremendous attention in scientific and academic circles as the pharmaceutical, commercial, and clinical potential of gene therapy has gradually emerged. The introduction of genes into cells of various origins, often referred to as gene transfection, is a critical component of any therapeutic or clinical regime.
Several different approaches have been developed for gene transfer. These include the use of viral based vectors (e.g., retroviruses, adenoviruses, and adeno-associated viruses) (Drumm, M. L. et al., Cell 62:1227-1233 (1990); Rosenfeld, M. A. et al., Cell 68:143-155 (1992); and Muzyczka, N., Curr. Top. Micro. Immuno. 158:97-129 (1992)), charge associating the DNA with an asialorosomucoid/poly L-lysine complex (Wilson, J. M. et al. (1992)), charge associating the DNA with cationic liposomes (Brigham, K. L. et al. (1993)) and the use of cationic liposomes in association with a poly-L-lysine antibody complex (Trubetskoy, V. S. et al., Biochem. Biophys. Acta 1131:311-313 (1993)).
To date, viral vectors have exhibited the highest levels of transfection efficiency for nucleic acids. Viral vectors have been particularly effective in in vivo systems, in contrast to non-viral vectors whose in vivo transfection efficiency has lagged. (Hanaria, E. G. et al., Am. Jnl. Microencapsulated. 99(5):537-52 (1995)) Although viral vectors are typically very effective at transfecting the cell, the use of viral vectors suffers from a major disadvantage. In particular, because the method infects an individual cell with a viral carrier, a potentially life threatening immune response to the treatment can develop. (Kingman, BioWorld Int., 1(20):1 (1996))
Although non-viral based transfection systems have not exhibited the efficiency of viral vectors, they have received significant attention, in both in vitro and in vivo research, because of their theoretical safety when compared to viral vectors. Synthetic cationic molecules, have been reported which reportedly "coat" the nucleic acid through the interaction of the cationic sites on the transfection agent and the anionic sites on the nucleic acid. The positively charged coating reportedly interacts with the negatively charged cell membrane to facilitate the passage of the nucleic acid through the cell membrane by non-specific endocytosis. (Schofield, Brit. Microencapsulated. Bull., 51(1):56-71 (1995)) These compounds have, however, exhibited considerable sensitivity to natural serum inhibition, which has probably limited their efficiency in vivo as gene transfection agents. (Behr, Bioconjugate Chem., 5, 382-389 (1994))
A number of attempts have been made to improve the efficiency of lipid-like cationic transfection agents, some involving the use of polycationic molecules. For example, several transfection agents have been developed that contain the polycationic compound spermine covalently attached to a lipid carrier. Behr, Bioconjugate Chem., 5, 382-389 (1994), discloses a lipopolyamine and shows it to be more efficient at transfecting cells than single charge molecules (albeit still less efficient than viral vectors). The agent reported by Behr was, however, toxic, and caused cell death.
Past attempts at nucleic acid transfection have also experienced difficulties with DNA precipitating out of solution. The problem is especially acute in in vivo applications wherein higher concentrations of DNA are often employed that create solubility problems for the DNA/carrier compound systems. Solutions to DNA precipitation have been addressed by research such as described in J. Bio. Chem. 271, no. 10 March 8, pp. 5656-5661. There, it was found that precipitation of DNA could be avoided by significantly stepping up the concentration of mono and multi-valent cations. While partly successful solubilizing the DNA/carrier complex, increasing ionic strength had toxic effects upon the transfected cells. Thus, there is a need in the art to enhance DNA uptake, in the presence of serum, which includes high DNA concentration levels in the nucleic acid/carrier system that are not prone to loss of solubility of DNA and without causing toxicity to the cells.
The present invention provides a new class of non-viral polymeric vectors that can be used for both in vitro and in vivo transfer of biologically active molecules. In particular, these vectors can be used for gene transfer applications. These polycationic non-lipid compounds can achieve gene transfer efficiencies in vitro that are superior to commercially available cationic liposome preparations. Further, their low toxicity and lack of serum inhibition is consistent with in vivo use. The present invention provides a vector that can achieve in vivo gene transfer efficiencies that compare favorably to viral vector systems. The present invention further provides a method to increase the capacity of solutions to carry complexes of nucleic acids and the polymeric vector without precipitation or toxic ionic effects on cells.
Furthermore, the unique polycationic structure of this class of polymers associates with many suitable bioactive molecules, including proteins and other compounds that possess multiple anionic sites. The polymer can act as a carrier to deliver the associated bioactive molecule, in vivo or in vitro, to the cells of interest for the bioactive molecule.